title: cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.
abstract: Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.
authors.affiliation: German Cancer Research Center, Division Biochemistry of the Cell (0225) and Biomedical Ultrastructure Research Unit (0195), D-69120 Heidelberg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Nehls
authors.forename: T
authors.initials: T
authors.lastname: Keck
authors.forename: R
authors.initials: R
authors.lastname: Greferath
authors.forename: E
authors.initials: E
authors.lastname: Spiess
authors.forename: T
authors.initials: T
authors.lastname: Glaser
authors.forename: K
authors.initials: K
authors.lastname: Rothbarth
authors.forename: H
authors.initials: H
authors.lastname: Stammer
authors.forename: D
authors.initials: D
authors.lastname: Werner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins